AMPA receptor properties and coexpression with sodium–calcium exchangers in rat hypothalamic neurons

Abstract

The histaminergic tuberomamillary (TM) nucleus, a center for the regulation of wakefulness, is excited by glutamatergic, aminergic and peptidergic inputs. AMPA receptor properties in relation to their expression were investigated in acutely isolated TM neurons with the help of whole-cell patch-clamp recordings combined with single-cell RT-PCR. The mRNAs encoding for the AMPA receptor GluR2 (100% of the neurons) and GluR1 (75%) were the most frequently detected, followed by the mRNA for GluR4 (56%), whereas GluR3 cDNA amplification did not yield a PCR product in any neuron. Flip splice variants prevailed over flop, in keeping with a strong glutamate-response potentiation by cyclothiazide. The expression pattern of AMPA subunits in their two splice variants was correlated with the different subtypes of Na+/Ca2+ (NCX) and Na+/Ca2+/K+ (NCKX) exchangers: glutamate receptor subunits GluR1-4 displayed no coordinated pattern with NCX. However, NCKX2 mRNA occurred only in TM cells with a fast desensitizing glutamate response, where it was coexpressed with the GluR4 subunit in the flop splice variant. NCKX3 mRNA was detected in neurons with fast or slow desensitization of glutamate responses. AMPA receptors in TM neurons were Ca2+-impermeable. As reverse Na+/Ca2+ exchange contributes to the immediate rise in intracellular calcium resulting from glutamate receptor activation, we suggest that the coordinated expression of NCKX2 with the fast desensitizing AMPA receptor-type reflects either a receptor-exchanger coupling or separate mechanisms for maintaining calcium homeostasis in neurons with fast or slow glutamate responses.

Editing of AMPA and Serotonin 2C Receptors in Individual Central Neurons, Controlling Wakefulness

“…It was shown previously that the prevailing expression of flop-splice variants of GluRs contributes to the faster desensitization of these receptors (Audinat et al 1996). In accordance, we have shown that GluR4-flop expression in TMN neurons coincides with the very fast desensitization of glutamate responses (Sergeeva et al 2004). Therefore, flop-expressing cells were excluded from the present study (n = 4).…”

“…Splice variants of AMPA receptors were analyzed by the restriction analysis (see Vorobjev et al 2000). In all investigated neurons the GluR4 subunit was present in the flip isoform (GluR4-flop expressing cells (see Sergeeva et al 2004) were rare and they were excluded from the study). The Q/R site-containing cDNA fragment was amplified in the second round with the Q/Rup: 5 0 -GGA AGA GAA ACA CAA AGT AGT G-3 0 and Q/Rlo: 5 0 -TTT GCT TAG ACA GAT CCT CAG C-3 0 primers.…”

“…All restriction endonucleases were obtained from New England Biolabs and Promega (USA). Negative controls were performed as previously described (Sergeeva et al 2004). Results are given as average ± SEM, the nonparametrical Wilcoxon and Mann-Whitney tests or the unpaired t-test were applied where appropriate for group comparison.…”

“…We have correlated ADAR-, 5-HT 2C R -and GluR-expression using the single-cell RT-PCR technique in individual neurons. We selected for this purpose hypothalamic histaminergic neurons acutely isolated from the tuberomamillary nucleus (TMN), which express GluR1, GluR2, and GluR4 AMPA receptor subunits (Sergeeva et al 2004) and the serotonin 2C receptor (Sergeeva et al 2003). As GluR1 subunit pre-mRNA does not undergo editing (Lomeli et al 1994), we determined the extent of editing at known sites of mRNAs encoding for the 5-HT 2C R, as well as GluR2 and GluR4 subunits of the AMPA receptor.…”

Neuropharmacology of Histamine in Brain

“…Serotonin depolarizes histamine neurons and increases their firing rate by activation of 5-HT 2C receptors and a NCX1 Na þ /Ca 2 þ exchanger [379,380]. Glutamatergic a-amino-3-hydroxy-5-methyl-4-isoxazole propinoic acid (AMPA) receptors coexpressed with NCKX Na þ /Ca 2 þ exchangers activate acutely isolated histamine neurons with different desensitization properties [381]. Although a purinergic transmission in the tuberomammillary nucleus has not yet been reported, adenosine triphosphate (ATP) can be coreleased with different transmitters such as GABA and excites individual histaminergic neurons through P2X receptors [382,383].…”